Multipotent Isl1+ cardiovascular precursors (MICPs) derived from post-natal and embryonic hearts have the capacity to differentiate into the critical cell lineages required for cardiogenesis: cardiomyocytes, smooth muscle and endothelial cells. Their identification in the adult heart has enormous implications for cardiac restorative function but little is known about these progenitors at the molecular level and how they are specified. We have recently developed a method whereby human embryonic stem cells (hESCs) can be uniformly differentiated into Isl1+ progenitors in chemically defined media. This represents an excellent opportunity to thoroughly understand the basic biology of Isl1+ progenitors and perhaps, to develop them as a potential source of cells that could eventually be used for cardiovascular cell therapies and tissue engineering. This proposal will: 1. characterize signaling pathways required for specification of hESC-derived Isl1+ progenitors. Since WntSa and BMP4 are required for this specification, special emphasis will be placed on their role in the generation of Isl1+ progenitors. 2. establish conditions for maintenance and amplification of Isl1+ progenitors. This will involve experiments where the molecular basis underpinning the Isl1+ state is investigated including analysis of signaling pathways and downstream transcription factor networks important for Isl1+ progenitor development. 3. establish the differentiation capacity of hESC-derived Isl1+ progenitors with the hypothesis that they have the potential to generate cardiomyocytes, smooth muscle cells and endothelial cells. In parallel, the ability of Isl1+ progenitors to function in animal model systems for cardiac and hind limb ischemia will be evaluated. The uniform, robust method for generation of Isl1+ progenitors that we have developed has the potential to significantly advance our understanding of early embryonic events associated with cardiac development and, for the development of cell therapeutics associated with cardiovascular disease.